Comprehensive Infectious Disease Diagnostic Protocols

We tested for Plasmodium spp, Leptospira spp, O tsutsugamushi, R typhi, spotted-fever-group rickettsia, causes of community bacteraemia, dengue fever, Japanese encephalitis virus, and, for the last 6 months at Luang Namtha, influenza (appendix). We did not test for tuberculosis or HIV. We followed manufacturer's instructions unless otherwise stated. We did Giemsa-stained malaria smears and plasmodium lactate-dehydrogenase-based immunochromatographic tests (ICT Malaria Combo Cassette Test; ICT Diagnostics, Cape Town, South Africa) for all patients. Full blood counts were done when possible at Salavan (ABX Micros 60 Hematology Analyzer, Horiba ABX, Japan) and Luang Namtha (Mindray BC 3000 Hematology Analyzer, Mindray Medical Instrumentation, NJ, USA). We identified positive blood cultures with conventional techniques³ (link) and antibiotic susceptibility by disc diffusion with Clinical and Laboratory Standards Institute criteria.¹⁴ We did rickettsial culture by inoculation of buffy coat onto Vero and L929 cells with incubation for 6–8 weeks, and speciation by immunofluorescence assay and PCR.¹⁵ (link) We undertook leptospiral culture with the clot remaining after centrifugation of clotted blood, with Ellinghausen-McCullough-Johnson-Harris medium.¹⁶ (link) Rickettsial and leptospiral culture began in August, 2009, representing 16 months of the study.
We used dengue and Japanese encephalitis virus ELISAs (Panbio, Brisbane, Australia) to detect dengue NS1, anti-dengue IgM and IgG, and anti-Japanese encephalitis virus IgM (appendix). Immunofluorescence assays were done for antibodies, in dried blood-spot elutes, against O tsutsugamushi and R typhi. We defined a positive result as an IgM or IgG titre of 1:400 or more.¹⁷ (link) We regarded leptospiral microscopic agglutination tests as positive if serum showed a titre of 1:400 or more or if paired sera showed a four-fold rise.⁹ (link)
We extracted nucleic acids and did all PCRs in duplicate on a Rotor-Gene 3000 or 6000 (Qiagen, Germany) for real-time PCR and a DNA Engine (MJ Research, Canada) for conventional PCR. We based detection of dengue virus on the single-step TaqMan real-time PCR assay.¹⁸ (link) For Plasmodium spp, we used a nested conventional PCR assay¹⁹ (link) targeting the ssrRNA gene, with distinguishing of P falciparum from P vivax. For Leptospira species, we used a TaqMan real-time PCR assay, detecting the Leptospira rrs gene.²⁰ (link)
We used three probe-based real-time PCR assays to detect O tsutsugamushi (47 kDa htrA gene), Rickettsia genus (17 kDa gene), and R typhi (ompB gene).21 (link), 22 (link), 23 (link), 24 (link) We regarded Rickettsia genus 17 kDa real-time PCR-positive samples, and R typhi ompB real-time PCR-negative samples as Rickettsia spp, which subsequently underwent a panel of nested conventional PCR assays targeting the 17 kDa, gltA, ompB, ompA, and sca4 genes.22 (link), 24 (link) For positive amplicons, DNA sequencing was done by Macrogen (Seoul, South Korea), followed by Basic Local Alignment Search Tool (BLAST) searches of GenBank. We collected nasopharyngeal or oropharyngeal swabs at Luang Namtha from June, 2010, to December, 2010. The National Centre for Laboratory and Epidemiology did influenza real-time PCR with US Centers for Disease Control and Prevention primers and probes for the influenza virus (H1N1, H3N2, pandemic H1N1 2009, H5N1, and influenza B).²⁵
We classified patients' diagnoses in two ways. First, the more conservative, and probably more accurate approach, using only diagnoses based on culture (ie, blood, rickettsial, and leptospiral culture), antigen detection (dengue NS1), and PCR (Plasmodium spp, O tsutsugamushi, R typhi, spotted-fever-group Rickettsia spp, Leptospira spp, and dengue) plus, potentially less reliably, anti-Japanese encephalitis virus IgM ELISA.²⁶ Second, we used all available tests (ie, the above plus O tsutsugamushi and R typhi immunofluorescence assay and dengue IgM and IgG ELISAs, which are likely to have lower specificity). Concordance between duplicate PCR assays was high (appendix), except for R typhi because of the difficulties in distinguishing R typhi from the spotted fever group. We analysed the association between patient symptoms, signs, and laboratory features for each aetiological diagnosis (see statistical analysis).